Process for preparing R-(−)-carnitin from S-(−)-chlorosuccinic acid or from a derivative thereof

ABSTRACT

An inner salt of L-carnitine is prepared by reduction, with a suitable reducing agent, of a compound of formula (I):                  
 
where X 1  and X 2 , which may be the same or different, are hydroxy, C 1 –C 4  alkoxy, phenoxy, halogen, or X 1  and X 2 , when taken together are an oxygen atom and the resulting compound is a derivative of succinic anhydride; Y is halogen, a mesyloxy or a tosyloxy group, and subsequent treatment with water, then with a base and then with trimethylamine.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 09/959,717filed Nov. 6, 2001 (allowed Sep. 3, 2003), now U.S. Pat. No. 6,677,476,which is a U.S. national phase of PCT/IT00/00187 filed May 12, 2000.

The invention described herein relates to a process for the preparationof R-(−)-carnitine (L-(−)-carnitine orR-(−)-3-hydroxy-4-(trimethylammonium)butyrate), hereinafter referred to,for the sake of brevity, as L-carnitine, starting fromS-(−)-chlorosuccinic acid or one of its derivatives.

BACKGROUND TO THE INVENTION

As is known, carnitine possesses an asymmetrical carbon atom and theenantiomer L-carnitine is the isomer present in living organisms, whereit is essential for fatty acid metabolism and functions actively in thetransport of fatty acids across the mitochondrial membranes. For thisreason L-carnitine, in addition to being a life-saving drug for thosewho suffer from an L-carnitine deficiency of genetic origin and to beingused in cases of temporary L-carnitine deficiency, such as, forinstance, those occurring after haemodialysis (U.S. Pat. No. 4,272,549,Sigma-Tau), plays an important role in energy metabolism and is regardedas a non-toxic natural product capable of enhancing cardiac function. Itis therefore used as a support drug in the treatment of various heartdiseases such as ischaemia, angina pectoris, arrhythmias, etc. (U.S.Pat. No. 4,649,159 and U.S. Pat. No. 4,656,191 Sigma-Tau). L-carnitineand its derivatives, moreover, have also been used to a significantextent as serum lipid lowering agents, anticonvulsants and blood productpreservatives. Recently, one of its derivatives, propionyl L-carnitine(Dromos®), was launched on the Italian market for the treatment ofintermittent claudication (U.S. Pat. No. 4,968,719, EP 0793962,Sigma-Tau).

There is also a substantially growing use of L-carnitine as a foodsupplement in the field of the so-called ‘health foods’ or“nutraceuticals”.

All this explains why L-carnitine is produced industrially in largeamounts and also why several attempts have been made to improve theindustrial synthesis of L-carnitine in terms of the cost of the product.

From a general point of view, the synthesis pathways that can be used tosynthesise L-carnitine are essentially three.

The first of these, starting from non-chiral or racemic compounds,passes through racemic intermediates, at the level of one of which theseparation of the useful enantiomer occurs, with methods known toexperts in pharmaceutical technology. Though this synthesis pathwaypresents the advantage of being able to rely on starting materials witha relatively low cost, for example, racemic carnitinamide (U.S. Pat. No.4,254,053, Sigma-Tau); racemic 2,3-dichloro-1-propanol (N. Kasai and K.Sakaguchi, Tetrahedron Lett. 1992, 33, 1211); 3-butenoic acid (D.Bianchi, W. Cabri, P. Cesti, F. Francalanci, M. Ricci, J. Org. Chem.,1988, 53, 104); racemic 3-chloro-2-hydroxy-trimethylammonium chloride(R. Voeffray, J. C. Perlberger, L. Tenud and J. Gosteli, Helv. Chim.Acta, 1987, 70, 2058); racemic epichlorohydrin (H. Löster and D. M.Müller, Wiss. Z. Karl-Marx-Univ. Leipzig Math.-Naturwiss. R. 1985, 34,212); diketene (L. Tenud, Lonza, DE 2,542,196, 2,542,227 and DE2,518,813), it also presents a serious drawback, in that, at the momentone wishes to isolate the useful enantiomer from a racemic mixture,there is a theoretical loss of at least 50% of the product on which saidseparation is operated. In practice, then, the yields in this synthesisstep are substantially lower (U.S. Pat. No. 4,254,053, Sigma-Tau) andthere is the drawback of having to recover the chiral compound used forthe separation of the racemic mixture.

The second synthesis pathway, again starting from non-chiral products,“creates” the chiral centre of the configuration desired, operating asynthesis step in a chiral environment, whether by means of a catalyst(H. C. Kolb, Y. L. Bennani and K. B. Sharpless, Tetrahedron: Asymmetry,1993, 4, 133; H. Takeda, S. Hosokawa, M. Aburatani and K. Achiwa,Synlett, 1991, 193; M. Kitamura, T. Ohkuma, H. Takaya and R. Noyori,Tetrahedron Lett., 1988, 29, 1555), or by means of an enzyme (U.S. Pat.No. 4,707,936, Lonza). The disadvantages of this pathway are the highcost of the catalysts and the fact that, at the time the chiral centreis created catalytically, one is normally unable to obtain the pureenantiomer, but mixtures are obtained with variable enantiomericexcesses of the useful isomer, with all the consequent difficulties ofhaving to separate two substances with the same physico-chemicalcharacteristics. In the case of the use of micro-organisms incontinuous-cycle reactors, the transformation of the starting productsinto end products is never complete and the end product has to bescrupulously purified of all organic impurities of cellular origin,which are dangerous in that they are potential allergens.

The third synthesis pathway involves the use of a chiral startingproduct, which is transformed into L-carnitine via a series of reactionswhich, if the chiral centre is affected, must be stereospecific, whichmeans that the stereochemistry of said centre must be maintained orcompletely inverted during the reaction, which is not always easy toachieve. If, on the other hand, the synthesis step does not affect thechiral centre, the enantiomeric excess (ee) of the end product must bethe same, or very close to, the starting product, which means that“racemising” reaction conditions must be carefully avoided. Anotherlimitation is the cost of the chiral starting products, which isnormally much higher than that of non-chiral products. The effect ofthese difficulties has been that none of the various processes startingfrom chiral products such as, for example, la R-(−)-epichlorohydrin (M.M. Kabat, A. R. Daniewski and W. Burger, Tetrahedron: Asymmetry, 1997,8, 2663); D-galactono-1,4-lactone (M. Bols, I. Lundt and C. Pedersen,Tetrahedron, 1992, 48, 319); R-(−)-malic acid (F. B. Bellamy, M.Bondoux, P. Dodey, Tetrahedron Lett. 1990, 31, 7323);R-(+)-4-chloro-3-hydroxybutyric acid (C. H. Wong, D. G. Drueckhammer andN. M. Sweers, J. Am. Chem. Soc., 1985, 107, 4028; D. Seebach, F.Giovannini and B. Lamatsch, Helv. Chim. Acta, 1985, 68, 958; E.Santaniello, R. Casati and F. Milani, J. Chem. Res., Synop., 1984, 132;B. Zhou, A. S. Gopalan, F. V. Middlesworth, W. R. Shieh and C. H. Sih;J. Am. Chem. Soc., 1983, 105, 5925); 4-hydroxy-L-proline (P. Renaud andD. Seebach, Synthesis, 1986, 424); (−)-β-pinene (R. Pellegata, I. Dosi,M. Villa, G. Lesma and G. Palmisano, Tetrahedron, 1985, 41, 5607);L-ascorbic acid or arabinose (K. Bock, I. Lundt and C. Pederson; ActaChem. Scand., Ser. B, 1983, 37, 341); D-mannitol (M. Fiorini and C.Valentini, Anic, EP 60.595), has to date been used for the industrialproduction of L-carnitine.

A case apart is the Sigma Tau Italian patent No. 1,256,705, which may beregarded as a mixture of the first and second synthesis pathways. Whatit describes, in fact, is the preparation of L-carnitine starting fromD-(+)-carnitine, obtained as a discard product from the L-carnitinepreparation process by resolution of the carnitinamide racemic mixtureby means of camphoric acid (U.S. Pat. No. 4,254,053, Sigma-Tau).

The bibliographical and patent references cited above merely give someidea of the vast body of work carried out in order to find aneconomically advantageous synthesis of L-carnitine. The fact is that theonly two processes which have proved industrially and economically validare those used by the two main manufacturers of L-carnitine, Sigma-Tauand Lonza, as described in the two above-mentioned patents, U.S. Pat.No. 4,254,053 and U.S. Pat. No. 4,708,936, which date back to 1978 and1987, respectively.

SUMMARY OF THE INVENTION

A process has now been found which starts from a chiral product andsolves all the problems of the “third pathway”, that is to say theproblem of the cost of the starting product and those of thestereospecificity and regiospecificity of the reactions necessary inorder to pass from S-(−)-chlorosuccminc acid, or one of its derivatives,to L-carnitine. The L-carnitine obtained is, in fact, particularly pure,with a D-carnitine percentage ≦0.2%.

In particular, the invention described herein relates to a process forthe preparation of L-carnitine inner salt which includes the reduction,with a suitable reducing agent, of a compound of formula (I)

where:

X₁ and X₂, which may be the same or different, are hydroxy, C₁–C₄alkoxy, phenoxy, halogen; or X₁ and X₂, when taken together are anoxygen atom and the resulting compound is a derivative of succinicanhydride;

Y is halogen, the mesyloxy or the tosyloxy group;

and subsequent treatment with a base and then with trimethylamine.

Examples of C₁–C₄ alkoxy groups are methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy and ter-butoxy. The methoxy and ethoxygroups are preferred. Examples of halogen are chlorine, bromine andiodine. Chlorine is preferred.

The reduction of the compound of formula (I) is done with a suitablereducing agent, which may be selected from those available by thosehaving ordinary experience in the field on the basis of their owngeneral knowledge of the sector. Reducing agents suitable forimplementing the process according to the invention described herein arehydrides. Examples of hydrides are diborane, mixed hydrides such aslithium and aluminimum hydride, lithium or sodium borohydride. Thechoice of a suitable reducing agent will be made in relation to thecompound of formula (I) to be treated. This choice is made by the personhaving ordinary experience in the field on the basis of his or hergeneral knowledge and no further explanation is necessary.

The process according to the invention is carried out in a suitablereaction medium, such as an organic solvent, preferably aprotic, forexample, tetrahydrofuran (THF), dioxane, ethylene glycol dimethyl ether(DME) or 2-methoxyethyl ether (Diglime).

The reaction temperature, reactant concentrations and all otherparameters useful for determining the reaction conditions can beobtained by consulting normal organic chemistry manuals.

In a first embodiment of the invention, the compound of formula (I) isS-(−)-chlorosuccinic acid (X₁ and X₂ are hydroxy and Y is chlorine).Said acid can be prepared with good yields and sterospecific reaction,e.g. from L-aspartic acid (S-(+)-aspartic acid) (J. A. Frick, J. B.Klassen, A. Bathe, J. M. Abramson and H. Rapoport, Synthesis, 1992, 7,621 and literature cited therein), or can be purchased on the market.

In this first embodiment, the reducing agent is diborane.

Carnitine inner salt is then obtained from the reduction product ofS-(−)-chlorosuccinic acid, without the isolation of any intermediateproduct, by treatment with aqueous sodium hydroxide and trimethylamine.The reaction temperature is not critical and can be convenientlyselected on the basis of the reaction medium chosen, the reactantconcentrations, and all other parameters useful for a successfulreaction exploitation. For example, the reaction can be conducted atroom temperature, but higher temperatures can also be used compatiblewith the reaction conditions.

In a second embodiment of the invention, the compound of formula (I) isthe one in which X₁ is hydroxy, X₂ is methoxy, and Y is a halogen,preferably chlorine. This preferred compound can be prepared, forexample, starting from S-(−)-chlorosuccinic acid, as seen above, bytransformation via the corresponding anhydride. Different2-halogen-substituted succinic acids are prepared according to knownmethods.

The conversion is achieved by treating the S-(−)-chlorosuccinic acidwith a dehydrating agent, preferably with acetyl chloride/acetic acid orwith acetic anhydride, at a temperature ranging from room temperature to90° C. Other modes of conversion, with other reactants, reaction mediaand conditions, which the expert technician can deduce from his or herown general knowledge, are also possible. The S-(−)-chlorosuccinicanhydride thus obtained is treated with a suitable amount of methanol toyield the compound of formula (I) desired. Compounds of formula (I) canbe obtained, according to variants of this second embodiment of theinvention, in which X₂ stands for one of the meanings envisaged, alkoxyor phenoxy, using suitable alcohol or phenol in the treatment of thestarting anhydride.

In this second embodiment, the reducing agent is a mixed hydride such aslithium borohydride or lithium and aluminium hydride.

Carnitine inner salt is in turn obtained directly from the reductionproduct of 1-methyl hydrogen (S)-2-chlorosuccinate without the isolationof any intermediate product, with aqueous sodium hydroxide andtrimethylanmine, in the same way as described for the first embodiment.

In a third embodiment of the invention, the compound of formula (I) isthe one in which X₁ and X₂ are a halogen, preferably chlorine, and Y isa halogen, preferably chlorine, and, more preferably X₁ and X₂ and Y arechlorine. S-(−)-chlorosuccinic acid dichloride can be prepared startingfrom S-(−)-chlorosuccinic acid with known reactions for obtaining acylchlorides. The other halogen derivatives envisaged in the invention canalso be prepared in the same way.

In this third embodiment, the preferred reducing agent is sodiumborohydride.

Carnitine inner salt in turn is obtained directly from the reductionproduct of the previous reaction in exactly the same way as in the casesdescribed above.

In a fourth embodiment of the invention, the compound of formula (I) isthe one in which X₁ and X₂ are hydroxy, and Y is the mesyloxy group.Said compound can be prepared starting from S-malic acid andmethanesulphonyl-chloride with known hydroxy acid functionalisationreactions. The compound of formula (I) in which Y is tosyloxy isprepared in the same way.

In this fourth embodiment, the reducing agent is diborane. Carnitineinner salt is then obtained from the reduction product of the previousreaction in exactly the same way as in the cases described above.

In a fifth embodiment of the invention, the compound of formula (I) isthe one in which X₁ and X₂ are methoxy and Y is a halogen, preferablychlorine. Said preferred compound can be prepared as described, forexample, in J. Am. Chem. Soc. (1952), 74, 3852–3856, starting fromS-(−)-chlorosuccinic acid and diazomethane or with methanol and acidcatalysis, preferably in the presence of dehydrating agents.

In this fifth embodiment, the preferred reducing agent is a mixedhydride such as lithium borohydride or lithium and aluminium hydride.

Carnitine inner salt is then obtained from the reduction product of theprevious reaction in exactly the same way as in the cases describedabove.

In a sixth embodiment of the invention, the compound of formula (I) isthe one in which X₁ and X₂ are taken together and are an oxygen atom,and Y is a halogen, or a mesyloxy or a tosyloxy, preferably a halogen.preferably chlorine.

This sixth embodiment shall be described in the foregoing in aparticularly detailed manner, being the preferred embodiment,comprising.

the transformation of S-(−)-chlorosuccinic acid into L-carnitine viaS-(−)-chlorosuccinic anhydride.

According to this embodiment, the process for the preparation ofL-carnitine inner salt includes the following steps:

a) transformation of S-(−)-chlorosuccinic acid into the correspondingS-(−)-chlorosuccinic anhydride;

b) reduction of S-(−)-chlorosuccinic anhydride with a mixed hydride, inthe presence of a solvent, obtaining a compound which, without beingisolated, is directly converted to L-carnitine inner salt by treatmentwith an alkaline hydroxide and trimethylamine.

The reaction diagram illustrating this process is the following:

BEST METHOD OF CARRYING OUT THE PROCESS ACCORDING TO THE INVENTION

Preparation of S-(−)-chlorosuccinic Acid

One of the main problems posed in synthesis processes at the industriallevel is the ratio of the costs of reactants and materials, such assolvents and auxiliary substances, to the yield of the end product.

In industrial chemistry, increasingly frequent use is being made ofchiral compounds, the procuring of which on the market in substantialamounts is adversely affected by the high costs and difficulties inpreparation.

S-(−)-chlorosuccinic acid is still by no means easy to procure on themarket, suggesting that it may be economically convenient to prepare itwithin the context of one's own synthesis processes where it is used asan intermediate.

In the reference cited above (J. A. Frick et al., 1992), the preparationof S-(−)-chlorosuccinic acid is simply described as: “S-aspartic acidwas converted to S-chlorosuccinic acid by treatment with sodium nitritein hydrochloric acid”. In the diagram on page 621 of the referencecited, the yield of the synthesis step from S-aspartic acid toS-chlorosuccinic acid is 70%. In the experimental part, the only exampleof preparation is provided for S-bromosuccinic acid, with a yield of88%. The bromosuccinic acid preparation conditions are not the same asthose described for chlorosuccinic acid, though they are taken as anexample by analogy. From the industrial point of view, the synthesis ofbromosuccinic acid described by Frick et al. is not very convenient ineconomic terms. In the first place, the dilutions of the reactionmixture are very high; by way of an example, S-(+)-aspartic acid ispresent to the extent of 5% w/v in the final reaction mixture. Adistinct disadvantage arises in the case of isolation of the end productby extraction, which requires a substantial amount of ethyl acetate inorder to obtain a 3% w/v solution of S-(−)-bromosuccinic acid. Inaddition, the end product, obtained with a stoichiometric yield of 88%,is not very pure, particularly as regards optical purity (e.e. =94%).

An improved process has now been found, in the course of development ofthe invention described herein, for the preparation ofS-(−)-chlorosuccinic acid, which makes it possible to achieve an atleast 80%, approximately, higher yield, better conditions of reaction,especially in terms of reaction volumes, and of product isolation, andre-use of reactants, with consequent savings in terms of industrialprocess costs.

Therefore, the framework of the invention described herein covers aprocess for the preparation of S-(−)-chlorosuccinic acid which includesthe reaction between S-(+)-aspartic acid and sodium nitrite in ahydrochloric acid-aqueous milieu, in the presence of sodium chloride,the improvement wherein consists in the isolation of the reactionproduct by precipitation by means of cooling of the reaction mixture.

Another object of the invention described herein is a process for thepreparation of S-(−)-chlorosuccinic acid which includes the reactionbetween S-(+)-aspartic acid and sodium nitrite in a hydrochloricacid-aqueous milieu, the improvement wherein consists in the use, as areaction medium, of the mother waters of a previous preparation reactionas in the process described above, said mother waters being used as atleast partial substitutes for the sodium chloride and hydrochloric acidenvisaged in the first process. According to this second process, thewashing waters of the end product of the previous reaction are also usedin addition to the mother waters.

The process for the preparation of S-(−)-chlorosuccinic acid accordingto the invention involves the reaction of S-(+)-aspartic acid-withsodium nitrite, in the presence of sodium chloride and concentratedhydrochloric acid.

The molar ratio of S-(+)-aspartic acid to sodium chloride ranges from1:0.3 to 1:0.5, preferably from 1:0.35 to 1:0.45. The precipitation isdone at a temperature ranging from −10° C. to −20° C., and preferably at−15° C.

According to the invention described herein, the concentration ofS-(+)-aspartic acid is greater than 15%, and preferably 16% w/v in thereaction mixture.

In a first embodiment of this process, S-(+)-aspartic acid is suspendedin demineralised water in a w/v ratio ranging from 1 kg/L to 0.5 kg/L,preferably 0.66 kg/L, in the presence of sodium chloride, in a molarratio as described above, and concentrated hydrochloric acid is added ina ratio of S-(+)-aspartic acid to hydrochloric acid ranging from 0.35kg/L to 0.55 kg/L, preferably 0.45 kg/L. The temperature of the mixtureis brought down below 0° C., preferably to −5° C. In a preferredembodiment of the process, the reaction mixture is protected in an inertatmosphere, e.g. nitrogen or argon. Sodium nitrite is then added inportions under stirring in a molar ratio ranging from 1.2 to 2.5,preferably 1.78. The sodium nitrite can be added in solid form ordissolved in a suitable amount of water. When the sodium nitrite isadded in the form of a solution, the latter is suitably prepared usingpart of the water initially envisaged for the aspartic acid suspension.Addition of the sodium nitrite is made by monitoring the reactiontemperature.

The reaction progress can be monitored by observing the development ofnitrogen. Once the reaction has begun, the development of nitrogen maysubstitute for the inert atmosphere mentioned above.

To facilitate completion of the reaction, when the addition of sodiumnitrite is completed, the reaction temperature can also be raised,either by leaving it to rise spontaneously or by heating the mixture.Preferably, the temperature should be brought up to 0° C.

The isolation of the product is done using conventional methods, but inthe context of the invention described herein, it has been found thatprecipitation of the end product by cooling, e.g. to −15° C., isparticularly advantageous, especially as regards the presence of organicimpurities in the reaction mixture (unreacted fumaric, malic andaspartic acids).

From the industrial point of view, the process according to theinvention is advantageous whether applied in successive cycles orcontinuously. In fact, successive preparations of S-(−)-chlorosuccinicacid allow part of the reactants to be recovered.

The mother waters and possibly also the washing waters of the firstreaction (REACTION A) are used as a reaction medium for a subsequentpreparation (REACTION B). Advantageously, the mother waters of REACTIONA (in actual fact, a brine) at low temperature (e.g. −15° C.) can bemixed with the other components of the following reaction (REACTION B),which leads to a frigorie saving and to a speeding-up of process times.Advantageously, in REACTION A, the reaction medium also includes thewashing waters of REACTION B. In REACTION A, the reaction medium canalso include the washing waters of REACTION B.

Thus, another object of the invention described herein is a process forthe preparation of S-(−)-chlorosuccinic acid which comprises thereaction between S-(+)-aspartic acid and sodium nitrite in ahydrochloric acid-aqueous milieu, the improvement wherein consists inthe use, as a reaction medium, of the mother waters of a previouspreparation reaction as described above, said mother waters being usedas at least partial substitutes for the sodium chloride and hydrochloricacid envisaged in the first reaction. Preferably, said mother waters areimmediately recycled at the S-(−)-chlorosuccinic acid precipitationtemperature envisaged for the previous reaction, so that, by mixing themwith the reactants still to be added, a temperature for the startingmixture around −5° C., being this latter the normal temperature for thisreaction, is immediately available. Advantageously, the washing watersfrom the previous reaction can also be used in addition to the motherwaters.

Alternatively, the process according to the invention includes thereaction between S-(+)-aspartic acid and sodium nitrite in ahydrochloric acid-aqueous milieu, the improvement wherein consists inthe use, as a reaction medium, of the mother waters of a previouspreparation reaction, said mother waters being used as at least partialsubstitutes for the sodium chloride and hydrochloric acid envisaged andsaid S-(−)-chlorosuccinic acid is isolated by extraction. In this case,a yield of over 90% is obtained, without any inorganic residue.

Preferably, said mother waters are immediately recycled at theS-(−)-chlorosuccinic acid precipitation temperature envisaged for theprevious reaction, so that, by mixing them with the reactants still tobe added, a temperature for the starting mixture around −5° C., beingthis latter the normal temperature for this reaction, is immediatelyavailable. The process according to the invention is even moreadvantageous if inserted in the context of the process for thepreparation of L-carnitine, which is the object of the inventiondescribed herein.

In fact, the S-(−)-chlorosuccinic acid, obtained according to theprecipitation method described herein contains a percentage of sodiumchloride ranging from 15 to 25%, but can be used directly for thepreparation of S-(−)-chlorosuccinic anhydride, where the sodium chloridecontent can be easily eliminated.

Therefore, a further object of the present invention is a process forthe preparation of S-(−)-chlorosuccinic anhydride which includes thereaction between S-(−)-chlorosuccinic acid and acetic anhydride, theimprovement wherein consists in the use of crude S-(−)-chlorosuccinicacid coming directly from the processes described above.

Preparation of S-(−)-chlorosuccinic Anhydride

S-(−)-chlorosuccinic anhydride, which is obtained fromS-(−)-chlorosuccinic acid by converting the bicarboxylic acid into ananhydride, is a new compound and therefore the invention describedherein includes said compound as a reaction intermediate in the processdescribed here. The conversion occurs by treating S-(−)-chlorosuccinicacid with a dehydrating agent, preferably acetyl chloride/acetic acid oracetic anhydride, at a temperature ranging from room temperature to 90°C.

The carnitine inner salt is in turn obtained from S-(−)-chlorosuccinicanhydride by reduction with a mixed hydride, preferably NaBH₄, in asuitable reaction medium, such as an organic solvent, preferablyaprotic, for example, tetrahydrofuran (THF), monoglyme, diglyme,dioxane, ethyl or methyl acetate (EtOAc or MeOAc) or a mixture of thesame, and by reaction of the crude product thus obtained with aqueoussodium hydroxide and trimethylamine at temperatures ranging from roomtemperature to 120° C., preferably from 60° C. to 100° C.

The compounds, 1-methyl hydrogen (S)-2-chlorosuccinate,(S)-2-chlorosuccinoyl dichloride and (S)-methanesulphonyloxysuccinicacid are new and are claimed herein as intermediates for the processaccording to the invention.

The L-carnitine inner salt can be salified with an acid, as indicatedschematically here below:

where X-^(⊖) is, for example, a halide ion (preferably chloride), anacid sulphate, a methane sulphonate or an acid fumarate,

or

where X^(2- ⊖) is the counter-ion of a bicarboxylic acid, such as, forexample, a tartrate ion or a mucate ion.

Of course, all possible salifications with suitable counter-ions arepossible, normally counter-ions of non-toxic acids, accepted forpharmaceutical, alimentary and livestock breeding uses, and for the usesenvisaged for L-carnitine and its derivatives, e.g. the acyl carnitines,carnitine esters and acyl carnitine esters.

The following examples further illustrate the invention describedherein.

EXAMPLE 1

S-(−)-chlorosuccinic Acid “REACTION A”

To a vigorously stirred mixture of 200 g (1.50 mol) of L-aspartic acid,40 g of sodium chloride (0.68 mol), 440 ml (523.6 g) of 37% HCl (193.74g of HCl, 5.32 mol), 200 ml of demineralised water, 100 ml of washingwaters of the solid obtained in “REACTION B” (see Example 2), are added184 g (2.66 mol) of solid sodium nitrite in approximately 2 hours at atemperature of −5° C. under nitrogen blanket. Stirring is continued atthe same temperature for 2.5 hours, the temperature is raised to +0° C.in the space of approximately 1 hour, the mixture is left at thistemperature for another period of 1 hour and then the temperature islowered to −15° C. After 1.5 hours at that temperature, the mixture isvacuum filtered on Buchner filters and left to “drain” under vacuum pumpaspiration for approximately 0.5 hours. The solid is then washed with 80ml of water at 0° C. and left on a vacuum filter for another 1.5 hours.

The crude product is vacuum dried in an oven at 40° C. It presentsapproximately 15–20% sodium chloride contamination.

The molar percentages of the impurities present, calculated on the basisof the NMR spectrum, are the following:

fumaric acid 0.1–0.2% w/w malic acid 0.1–0.4% w/w aspartic acid 0.1–0.2%w/w

The yield of S-(−)-chlorosuccinic acid, calculated 100% pure, is 80–81%.

EXAMPLE 2

S-(−)-chlorosuccinic Acid “REACTION B”

To a vigorously stirred mixture of mother waters and washing waters(approximately 650 ml) from the previous reaction are added 200 g (1.50mol) of L-aspartic acid, 360 ml (428.4 g) of 37% HCl (158.51 g of HCl,4.35 mol) and 100 ml of demineralised water; 184 g (2.66 mol) of solidsodium nitrite are then added in approximately 2 hours at a temperatureof −5° C. under nitrogen blanket. Stirring is continued at the sametemperature for 2.5 hours, the temperature is raised to +0° C. in thespace of approximately 1 hour, the mixture is left at this temperaturefor another period of 1 hour, and the temperature then lowered to −15°C. After 1.5 hours at this temperature, the mixture is vacuum filteredon Buchner filters and left to “drain” under vacuum pump aspiration forapproximately 0.5 hours. The solid is then washed with 80 ml of water at0° C. and left on a vacuum filter for another 1.5 hours.

The crude product is vacuum dried in an oven at 40° C. It presentsapproximately 15–20% sodium chloride contamination.

The molar percentages of the impurities present, calculated on the basisof the NMR spectrum, are the following:

fumaric acid 0.1–0.2% w/w malic acid 0.1–0.4% w/w aspartic acid 0.1–0.2%w/w

The yield of S-(−)-chlorosuccinic acid, calculated 100% pure, is 86–87%.

The pure product, obtained by means of a further crystallisation of asample of the crude product with water, has a melting point of 180–182°C.

The overall yield of reactions A(+)B is 83–84%.

EXAMPLE 3

S-(−)-chlorosuccinic Anhydride

A suspension of 300 g (1.97 mol) of S-(−)-chlorosuccinic acid,containing 45–80 g of sodium chloride as a residue of the previouspreparation and 241.5 mL (2.56 mol) of acetic anhydride is stirred at52–55° C. for 3.5 hours. The insoluble sodium chloride is filtered outand the clear, colourless solution is vacuum evaporated and dried. Toeliminate the last residues of acetic acid and acetic anhydride, thesolid residue is extracted with 300 ml of anhydrous isopropyl ether, thesuspension is stirred vigorously for 5 minutes and filtered, and thesolid is washed on the filter with another 90 ml (66 g) of freshisopropyl ether. After vacuum drying in an anhydrous milieu, 251.3 g ofS-(−)-chlorosuccinic anhydride are obtained (95%; m.p. 75–80° C.;[α]D=−4.16 (c=1.0; ethyl acetate)).

EXAMPLE 4

S-(−)-chlorosuccinic Anhydride

A suspension of 53 g (0.347 mol) of S-(−)-chlorosuccinic acid in 38 mL(0.40 mol) of acetic anhydride was stirred at 70° C. until the solid wascompletely dissolved, after which the acetic acid and excess aceticanhydride were vacuum distilled. At this point S-(−)-chlorosuccinicanhydride could be recovered by filtration, after treatment withcyclohexane, or by distillation at 0.5 mm Hg. Yields of around 95% wereobtained in all cases (=44.4 g) (ee≧99%).

Elemental Analysis for: C₄ H₃ Cl O₃ C % H % Cl % Calc. 35.72 2.25 26.36Found 35.62 2.20 26.21 [α]D²⁵ = −3.78° (c = 10, EtOAc)

¹H NMR (CDCl₃, δ, p.p.m.): 3.21 (dd, J=18.7), 5.2, (1H, CHH—CO); 3.59(dd, J=18.7), 9.0, (1H, CHH—CO); 4.86 (dd, J=9.0, 5.2, 1H, CH—Cl);

EXAMPLE 5

1-Methylhydrogen (S)-2-chlorosuccinate

To a solution of 6.00 g (0.0446 mol) of (S)-chlorosuccinic anhydride in60 mL of CHCl₃, without ethanol, held at −65 C, was added slowly amixture of 1.80 mL (0.0446 mol) of MeOH in 20 mL of CHCl₃. The solutionwas maintained at the same temperature for 1 hour and then left to riseto room temperature in 3 hours. After another 2 hours, the solution waswashed with 10 mL of NaOH 1N, dried on anhydrous sodium sulphate andvacuum evaporated to dryness. After purification on a chromatographiccolumn, 5.94 g (80%) of the title compound were obtained. H-NMR inDMSO-d₆: δ 2.89 (1H, dd, CHHCHCl), 3.00 (1H, dd, CHHCHCl), 3.71 (3H, s,COOCH₃), 4.78 (1H, t, CHCl).

EXAMPLE 6

(S)-2-chlorosuccinoyldichloride

A suspension of 10.00 g (0.0656 mol) of (S)-chlorosuccinic acid in 20.0mL (0.274 mol) of thionyl chloride was refluxed for 1 hour. Aftercooling, the solution was vacuum evaporated to dryness. The residue wasdistilled at 90–93C/10 mm Hg to obtain 12.56 g (85%) of the titlecompound. H-NMR in DMSO-d₆: δ 3.50 (1H, dd, CHHCHCl), 3.60 (1H, dd,CHHCHCl), 5.20 (1H, t, CHCl).

EXAMPLE 7

(S)-methane-sulphonyloxysuccinic Acid

A solution of 8.04 g (0.060 mol) of (S)-malic acid and 9.2 mL (0.120mol) of methanesulphonyl chloride in 60.0 mL of THF was refluxed for 10hours. After cooling, the solution was vacuum evaporated to dryness toobtain 12.60 g (99%) of the title compound. H-NMR in DMSO-d₆: δ 2.41(3H, s, CH₃SO₃), 2.90 (2H, m, CH₂), 5.47 (1H, t, CHOSO₂).

EXAMPLE 8

Dimethyl (S)-chlorosuccinate

To a solution of 7.04 g (0.046 mol) of (S)-chlorosuccinic acid in 60 mLof methanol were added 2.0 mL of concentrated H₂SO₄. After 3 days atroom temperature the solution was vacuum evaporated and the residueextracted with EtOAc. The solution was washed with a 5% NaHCO₃ aqueoussolution and the organic phase was dried on Na₂SO₄. 7.90 g (94%) of thetitle compound were obtained by evaporation. H-NMR in DMSO-d₆: δ 3.00(1H, dd, CHHCHCl), 3.12 (1H, dd, CHHCHCl), 3.61 (3H, s, COOCH₃), 3.71(3H, s, COOCH₃), 4.77 (1H, t, CHCl).

EXAMPLE 9

L-carnitine Inner Salt by Reduction of (S)-2-chlorosuccinic Acid

To a suspension of 6.00 g (0.039 mol) of (S)-chlorosuccinic acid in 20mL of anhydrous THF maintained at −15 C under nitrogen were added 58.5mL (0.0585 mol) of a 1M solution of borane in THF in 2 hours. After 20hours at the same temperature, the mixture was treated with 5.5 mL ofwater and left to stir at room temperature for 3 hours. After theaddition of 11 mL of 6M NaOH, the phases were separated. To the aqueousphase were added 7 mL of 40% Me₃N in water and the solution was left tostir at room temperature for 3 hours. The solution was vacuumconcentrated and the resulting solution brought to pH 5 with 37% HCl. Bymeans of evaporation of this solution a solid was obtained which wasextracted with 30 ml of MeOH. The solution obtained by filtration of theinsoluble part was vacuum evaporated and dried. The crude product waspurified on an ion-exchange column (Amberlite IR 120 form H⁺) by elutionwith 2% NH₄OH. By means of evaporation of the fractions containing thepure product, 3.14 g (50%) of L-carnitine were obtained.

EXAMPLE 10

L-carnitine Inner Salt by Reduction of 1-methyl hydrogen(S)-2-chlorosuccinate

To a suspension of 6.50 g (0.039 mol) of methyl (S)-2-chlorosuccinate in30 mL of anhydrous DME held at −15C under nitrogen were added 0.87 g(0.040 mol) of 95% LiBH₄ in portions in 2 hours. After 20 hours at thesame temperature the mixture was treated as described in Example 9 aboveto obtain 3.45 g (55%) of L-carnitine.

EXAMPLE 11

L-carnitine Inner Salt by Reduction of (S)-2-chlorosuccinoyl Dichloride

To a solution of 7.39 g (0.039 mol) of (S)-2-chlorosuccinoyl-dichloridein 30 mL of anhydrous DME held at −15C under nitrogen were added 0.74 g(0.0195 mol) of NaBH₄ in portions in 2 hours. After 20 hours at the sametemperature the mixture was treated as described in example 9 to obtain2.83 g (45%) of L-carnitine.

EXAMPLE 12

L-carnitine Inner Salt by Reduction of (S)-2-methanesulphonyloxysuccinicAcid

To a suspension of 8.27 g (0.039 mol) of (S)-methanesulphonyloxysuccinicacid in 30 mL of anhydrous THF held at −15C under nitrogen were added58.5 mL (0.0585 mol) of a 1M solution of borane in THF in 2 hours. After20 hours at the same temperature, the mixture was treated as describedin Example 9 to obtain 2.51 g (40%) of L-carnitine.

EXAMPLE 13

L-carnitine Inner Salt by Reduction of dimethyl (S)-2-chlorosuccinate

To a suspension of 7.04 g (0.039 mol) of dimethyl (S)-2-chlorosuccinatein 30 mL of anhydrous DME held at −15C under nitrogen, were added 0.69 g(0.030 mol) of 95% LiBH₄ in portions in 2 hours. After 20 hours at thesame temperature the mixture was treated as described in Example 9 toobtain 3.32 g (53%) of L-carnitine.

EXAMPLE 14

S-(−)-chlorosuccinic Anhydride

A suspension of 53 g (0.347 mol) of S-(−)-chlorosuccinic acid in 38 mL(0.40 mol) of acetic anhydride was stirred at 70° C. until the solid hadcompletely dissolved, after which the acetic acid and excess aceticanhydride were vacuum distilled. At this point, the S-(−)-chlorosuccinicanhydride could be recovered by filtration after treatment withcyclohexane, or by distillation at 0.5 mm Hg. Yields of around 95%(=44.4 g) were obtained in all cases. (ee≧99%).

Elemental Analysis for: C₄ H₃ Cl O₃ C % H % Cl % Calc. 35.72 2.25 26.36Found 35.62 2.20 26.21 [α]D²⁵ = −3.78° (c = 10, EtOAc)

¹H NMR (CDCl₃, δ, p.p.m.): 4.86 (dd, J=9.0 Hz, 5.2 Hz, 1H, CH-Cl); 3.59(dd, J=18.7 Hz, 9.0 Hz, 1H, CHH—CO); 3.21 (dd, J=18.7 Hz, 5.2 Hz, 1H,CHH—CO)

L-carnitine Inner Salt

To a vigorously stirred suspension of 6.13 g (0.162 mol) of NaBH₄ in 18mL of anhydrous THF, held at 0° C., were added 43.4 g (0.323 mol) ofS-(−)chlorosuccinic anhydride in 90 mL of anhydrous THF. Thesuspension/solution was stirred for 8 hours at that temperature, thenquenched with water, left to stir for one hour and then added with NaOH4N in two portions, the first to bring the suspension to pH 7.5 and thesecond, after vacuum evaporating the organic solvent, to ensure totaladdition of 0.484 mol of NaOH (in all, 121 mL). To said solution wereadded 51 mL (0.337 mol) of a 40% aqueous solution of Me₃N, and the wholewas transferred into a closed vessel and held for 16 hours at 70° C. Atthe end of the reaction, the residual trimethylamine was eliminated byvacuum evaporation and then 80.75 mL (0.323 mol) of HCl 4N were added.The solution, containing L-carnitine inner salt, together withapproximately 8% of impurities (mainly fumaric acid, maleic acid,hydroxycrotonic acid, D-carnitine) and sodium chloride, was-desalted byelectrodialysis and then vacuum dried. 38.5 g of a crude product wereobtained which was crystallised with isobutylic alcohol to yield 31.4 g(60.4%) of pure L-carnitine inner salt. (ee≧99.6%).

Elemental Analysis for: C₇ H₁₅ N O₃ C % H % N % Calc. 52.16 9.38 8.69Found 52.00 9.44 8.59 [α]D²⁵ = −31.1° (c = 1.0, H₂O)

¹H NMR (D₂O, δ, p.p.m.): 4.57 (m, 1H, CH—O); 3.41 (d, 2H, CH₂—COO); 3.24(s, 9H, (CH₃)₃—N); 2.45 (d, 2H, CH₂—N)

L-carnitine Chloride

The reaction was repeated exactly as described above, except that, atthe end of the reaction in a closed vessel, the contents after coolingwere vacuum-dried. The residue was extracted with 53.5 ml (0.646 mol) of37% HCl and vacuum dried again. The residue was extracted twice withethanol; the first time with 200 mL and the second time with 60 mL,settling/filtering both times. The pooled ethanol solutions werevacuum-concentrated to a volume of approximately 50 mL, to which wereadded 600 mL of acetone to precipitate L-carnitine chloride. After onenight at room temperature the solid was filtered to yield 47.8 g ofcrude L-carnitine chloride. 38.5 g (60.4%) of pure L-carnitine chloridewere obtained by crystallisation with isopropanol (ee≧99.6%).

Elemental Analysis for C₇ H₁₆ Cl N O₃ C % H % Cl % N % Calc. 42.54 8.1617.94 7.09 Found 42.40 8.12 18.00 7.05 [α]D²⁵ = −23.0° (c = 0.86, H₂O)

¹H NMR (CD₃OD, δ, p.p.m.): 4.58 (m, 1H, CH—O); 3.48 (m, 2H, CH₂—N); 3.27(s, (CH₃)₃—N); 2.56 (d, J=6.7 Hz, 2H, CH₂—COOH)

1. A process for the preparation of S-(−)-chlorosuccinic anhydridecomprising a) reacting S-(+)-aspartic acid and sodium nitrite in ahydrochloric acid-aqueous milieu in which said S-(+)-aspartic acidsuspended in demineralized water in a w/w ratio ranging from 1 kg/L to0.5 kg/L, and concentrated hydrochloric acid is added in a ratio ofS-(+)-aspartic acid to hydrochloric acid ranging from 0.35 kg/L to 0.55kg/L in the presence of sodium chloride, said S-(+)-aspartic acid andsaid sodium chloride being in a molar ratio ranging from 1:0.3 to 1:0.5,to give crude S-(−)-chlorosuccinic acid; b) isolating said crudeS-(−)-chlorosuccinic acid by precipitation by cooling the reactionmixture at a temperature ranging from−10° C. to−20° C.; and c) reactingsaid isolated crude S-(−)-chlorosuccinic acid and acetic anhydride.